A common genetic alteration in glioblastoma multiforme (GBM) which confers aggressive tumor growth and poor prognosis is deletional mutation of the epidermal growth factor receptor (EGFR) resulting in a constitutively active, oncogenic form of the protein (EGFR*). However, GBM patients treated with drugs which selectively target EGFR/EGFR* rarely, if ever, benefit from therapy. Moreover, patients which initially respond almost always gain resistance and exhibit rapid disease progression. The proposed project aims to define mechanisms involved in resistance to EGFR inhibition with the overall goal of shedding light on those specific pathways and proteins involved which can be exploited therapeutically in combination with EGFR inhibitors. The project will utilize data obtained from an inducible mouse model of EGFR*-dependent GBM tumor formation. Following silencing of EGFR* expression, tumors regressed, and after a period of stasis, EGFR*- independent "escapers" emerged. These escapers will be characterized compared to EGFR*-dependent tumors in an effort to uncover those pathways and proteins utilized after cells in vivo circumvent the requirement for EGFR* signaling, which is similar to the situation in which GBM tumors do not clinically respond to EGFR inhibition for reasons of either acquired resistance or lack of initial dependence on the receptor. Specifically, Aim1 will examine the mechanisms of EGFR-independent signaling and resistance to tyrosine kinase inhibitors (TKIs) in the context of signaling pathway networks by manipulation of component protein expression and activation/phosphorylation. In Aim 2, a functional characterization of novel mechanisms that lead to escape and resistance will be performed using existing gene expression data as a starting point and defined elements for triaging candidate genes. Aim 3 comprises the development of therapeutic targeting strategies based on the findings in Aims 1 and 2. The goal of these targeting strategies is to overcome and/or prevent resistance to EGFR inhibition, and these studies are based largely upon use of gene expression and other data from trials in which patients were treated with TKIs. Overall, the knowledge gained from this project will shed light on the molecular basis of glioma pathobiology, provide potential ways to circumvent acquired resistance to EGFR inhibitors, and will reveal novel targeting strategies for anti-GBM therapeutics. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM), a grade IV astrocytoma and the most common primary malignant brain tumor in adults, has proven to be largely resistant to the multiple treatment approaches. This project aims to gain a greater understanding of the mechanisms involved in overcoming dependence on mutant epidermal growth factor receptor signaling and clinical resistance to therapeutic receptor inhibition and to provide a rationale for novel therapeutic approaches. As a result, strategies for the clinical management of this disease may be impacted in such a way that treatment of patients includes a combination of agents that are more well-suited to arrest the disease process at a molecular level to achieve improved clinical outcome.